The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a complex enzyme system which in Salmonella typhimurium and Escherichia coli catalyzes the concomitant transmembrane transport and phosphorylation of its sugar substrates. It also regulates the utilization of a variety of carbon sources not taken up via the PTS-catalyzed group translocation mechanism. Recent studies in our laboratory have provided new information regarding the mechanisms by which the PTS functions in catalysis and regulation of sugar uptake. The purpose of the proposed research is to test our current hypotheses and to establish novel regulatory mechanisms. Both biochemical and genetic approaches will be taken. A. Mechanism of Enzyme IIMt1 catalyzed transmembrane sugar transport. Studies will be undertaken to determine the mchanisms of the reactions catalyzed by homogeneous Enzyme IIMt1. Reconstitution of transport function in artificial liposomes will be attempted. Biogensis of the enzyme will be investigated employing a minicell strain carrying a plasmid coding for the mtl operon. B. Mechanisms of PTS-mediated regulation of carbon utilization. Known regulatory mutations (lac, crrA, crrB, crrC) will be studied, and novel regulatory loci will be sought. Our biochemical studies on transport regulation in membrane vesicles will continue, and an attempt will be made to extend studies with the vesicular system to adenylate cyclase. C. Evolutionary relatedness of PTS proteins. Immunological cross reactivities, substrate specificities, and susceptibilities of the Enzyme II complexes to protein reagents will be used to estimate the relatedness of the various Enzyme II complexes in E. coli and other bacteria. Phosphotransferase systems will be sought in selected bacterial groupings and in eukaryotic anaerobes.